Antibacterial agents

ABSTRACT

Compounds of formula (I) are antibacterial agents wherein: R 3  and R 4 , taken together with the carbon atoms to which they are respectively attached, form an optionally substituted saturated carbocyclic or heterocyclic ring of 5 to 16 atoms, which may be benz-fused or fused to a second optionally substituted saturated carbocyclic or heterocyclic ring of 5 to 16 atoms; and R 1  and R 2  are as defined in the specification.

This invention relates to a novel class of N-formyl hydroxylaminederivatives having antibacterial activity, and to pharmaceutical andveterinary compositions comprising such compounds.

BACKGROUND OF THE INVENTION

In general, bacterial pathogens are classified as either Gram-positiveor Gram-negative. Many antibacterial agents (including antibiotics) arespecific against one or other Gram-class of pathogens. Antibacterialagents effective against both Gram-positive and Gram-negative pathogensare therefore generally regarded as having broad-spectrum activity.

Many classes of antibacterial agents are known, including thepenicillins and cephalosporins, tetracyclines, sulfonamides,monobactams, fluoroquinolones and quinolones, aminoglycosides,glycopeptides, macrolides, polymyxins, lincosamides, trimethoprim andchloramphenicol. The fundamental mechanisms of action of theseantibacterial classes vary.

Bacterial resistance to many known antibacterials is a growing problem.Accordingly there is a continuing need in the art for alternativeantibacterial agents, especially those which have mechanisms of actionfundamentally different from the known classes.

Amongst the Gram-positive pathogens, such as Staphylococci,Streptococci, Mycobacteria and Enterococci, resistant strains haveevolved/arisen which makes them particularly difficult to eradicate.Examples of such strains are methicillin resistant Staphylococcus aureus(MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS),penicillin resistant Streptococcus pneumoniae and multiply resistantEnterococcus faecium.

Pathogenic bacteria are often resistant to the aminoglycoside, β-lactam(penicillins and cephalosporins), and chloramphenicol types ofantibiotic. This resistance involves the enzymatic inactivation of theantibiotic by hydrolysis or by formation of inactive derivatives. Theβ-lactam (penicillin and cephalosporin) family of antibiotics arecharacterised by the presence of a β-lactam ring structure. Resistanceto this family of antibiotics in clinical isolates is most commonly dueto the production of a “penicillinase” (β-lactamase) enzyme by theresistant bacterium which hydrolyses the β-lactam ring thus eliminatingits antibacterial activity.

Recently there has been an emergence of vancomycin-resistant strains ofenterococci (Woodford N. 1998 Glycopeptide-resistant enterococci: adecade of experience. Journal of Medical Microbiology. 47(10):849-62).Vancomycin-resistant enterococci are particularly hazardous in that theyare frequent causes of hospital based infections and are inherentlyresistant to most antibiotics. Vancomycin works by binding to theterminal D-Ala-D-Ala residues of the cell wall peptidioglycan precursor.The high-level resistance to vancomycin is known as VanA and isconferred by a genes located on a transposable element which alter theterminal residues to D-Ala-D-lac thus reducing the affinity forvancomycin.

In view of the rapid emergence of multidrug-resistant bacteria, thedevelopment of antibacterial agents with novel modes of action that areeffective against the growing number of resistant bacteria, particularlythe vancomycin resistant enterococci and β-lactam antibiotic-resistantbacteria, such as methicillin-resistant Staphylococcus aureus, is ofutmost importance.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the finding that certain N-formylhydroxylamine derivatives have antibacterial activity, and makesavailable a new class of antibacterial agents.

Although it may be of interest to establish the mechanism of action ofthe compounds with which the invention is concerned, it is their abilityto inhibit bacterial growth, which makes them useful. However, it ispresently believed that their antibacterial activity is due, at least inpart, to intracellular inhibition of bacterial polypeptide deformylase(PDF) enzyme.

Bacterial polypeptide deformylases (PDF) (EC 3.5.1.31), are a conservedfamily of metalloenzymes (Reviewed: Meinnel T, Lazennec C, Villoing S,Blanquet S, 1997, Journal of Molecular Biology 267, 749-761) which areessential for bacterial viability, their function being to remove theformyl group from the N-terminal methionine residue ofribosome-synthesised proteins in eubacteria. Mazel et al. (EMBO J.13(4):914-923, 1994) have recently cloned and characterised an E. coliPDF. As PDF is essential to the growth of bacteria and there is noeukaryotic counterpart to PDF, Mazel et al. (ibid), Rajagopalan et al.(J. Am. Chem. Soc. 119:12418-12419, 1997) and Beckeretal., (J. BiolChem. 273(19):11413-11416, 1998) have each proposed that PDF is anexcellent anti-bacterial target.

Certain N-formyl hydroxylamine derivatives have previously been claimedin the patent publications listed below, although very few examples ofsuch compounds have been specifically made and described:

EP-B-0236872 (Roche)

WO 92/09563 (Glycomed)

WO 92/04735 (Syntex)

WO 95/19965 (Glycomed)

WO 95/22966 (Sanofi Winthrop)

WO 95/33709 (Roche)

WO 96/23791 (Syntex)

WO 96/16027 (Syntex/Agouron)

WO 97/03783 (British Biotech)

WO 97/18207 (DuPont Merck)

WO 98/38179 (GlaxoWelicome)

WO 98/47863 (Labs Jaques Logeais)

The pharmaceutical utility ascribed to the N-formyl hydroxylaminederivatives in those publications is the ability to inhibit matrixmetalloproteinases (MMPs) and in some cases release of tumour necrosisfactor (TNF), and hence the treatment of diseases or conditions mediatedby those enzymes, such as cancer and rheumatoid arthritis. That priorart does not disclose or imply that N-formyl hydroxylamine derivativeshave antibacterial activity.

In addition to these, U.S. Pat. No. 4,738,803 (Roques et al.) alsodiscloses N-formyl hydroxylamine derivatives, however, these compoundsare disclosed as enkephalinase inhibitors and are proposed for use asantidepressants and hypotensive agents. Also, WO 97/38705 (Bristol-MyersSquibb) discloses certain N-formyl hydroxylamine derivatives asenkephalinase and angiotensin converting enzyme inhibitors. This priorart does not disclose or imply that N-formyl hydroxylamine derivativeshave antibacterial activity either.

Our copending International patent application no. PCT/GB99/00386discloses that certain N-formyl hydroxylamine derivatives haveantibacterial activity. One class of compounds disclosed as having suchactivity has general formula (IA):

wherein the various “R” substituents are as specified in the document.The compounds useful in accordance with the present invention differ instructure from those of PCT/GB99/00386 principally in that the acyclicamidoalkyl radical shown as lying to the right of the curved line informula (IA) is replaced by a cyclic radical.

DETAILED DESCRIPTION IF THE INVENTION

According to the first aspect of the present invention there is provideda compound of formula (I) or a pharmaceutically or veterinarilyacceptable salt, hydrate or solvate thereof:

wherein:

R₁ represents hydrogen, methyl, or trifluoromethyl

R₂ represents a group R₁₀-(X)_(n)-(ALK)- wherein

Rl, represents hydrogen, a C₁-C₆ alkyl, C₂-C alkenyl, C₂-C₆ alkynyl,cycloalkyl, aryl, or heterocyclyl group, any of which may beunsubstituted or substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy,mercapto, (C₁-C₆)alkylthio, amino, halo (including fluoro, chloro, bromoand iodo), trifluoromethyl, cyano, nitro, -COOH, -CONH₂, -COOR^(A),-NHCOR^(A), -CONHR^(A), -NHR^(A), -NR^(A)R, or -CONR^(A)R^(B) whereinR^(A) and R^(B) are independently a (C₁-C₆)alkyl group, and

ALK represents a straight or branched divalent C₁-C₆ alkylene, C₂-C₆alkenylene, C₂-C₆ alkynylene radical, and may be interrupted by one ormore non-adjacent —NH—, —O— or —S— linkages,

X represents —NH—, —O— or —S—, and

n is 0 or 1; and

R₃ and R₄, taken together with the carbon atoms to which they arerespectively attached, form an optionally substituted saturatedcarbocyclic or heterocyclic ring of 5 to 16 atoms, which may bebenz-fused or fused to a second optionally substituted saturatedcarbocyclic or heterocyclic ring of 5 to 16 atoms.

In another aspect, the invention provides the use of a compound offormula (I) as defined above in the preparation of a composition fortreatment of bacterial infections in humans and non-human mammals.

In another aspect, the invention provides a method for the treatment ofbacterial infections in humans and non-human mammals, which comprisesadministering to a subject suffering such infection an antibacteriallyeffective dose of a compound of formula (I) as defined above.

In a further aspect of the invention there is provided a method for thetreatment of bacterial contamination by applying an antibacteriallyeffective amount of a compound of formula (I) as defined above to thesite of contamination.

The compounds of formula (I) as defined above may be used ascomponent(s) of antibacterial cleaning or disinfecting materials.

On the hypothesis that the compounds (I) act by inhibition ofintracellular PDF, the most potent antibacterial effect may be achievedby using compounds which efficiently pass through the bacterial cellwall. Thus, compounds which are highly active as inhibitors of PDF invitro and which penetrate bacterial cells are preferred for use inaccordance with the invention. It is to be expected that theantibacterial potency of compounds which are potent inhibitors of thePDF enzyme in vitro, but are poorly cell penetrant, may be improved bytheir use in the form of a prodrug, ie a structurally modified analoguewhich is converted to the parent molecule of formula (I), for example byenzymic action, after it has passed through the bacterial cell wall.

As used herein the term “(C₁-C₆)alkyl” means a straight or branchedchain alkyl moiety having from 1 to 6 carbon atoms, including forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term “divalent (C₁-C₆)alkylene radical” means asaturated hydrocarbon chain having from 1 to 6 carbon atoms and twounsatisfied valencies.

As used herein the term “(C₂-C₆)alkenyl” means a straight or branchedchain alkenyl moiety having from 2 to 6 carbon atoms having at least onedouble bond of either E or Z stereochemistry where applicable. The termincludes, for example, vinyl, allyl, 1- and 2-butenyl and2-methyl-2-propenyl.

As used herein the term “divalent (C₂-C₆)alkenylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one doublebond, and two unsatisfied valencies.

As used herein the term “C₂-C₆ alkynyl” refers to straight chain orbranched chain hydrocarbon groups having from two to six carbon atomsand having in addition one triple bond. This term would include forexample, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and5-hexynyl.

As used herein the term “divalent (C₂-C₆)alkynylene radical” means ahydrocarbon chain having from 2 to 6 carbon atoms, at least one triplebond, and two unsatisfied valencies.

As used herein the term “cycloalkyl” means a saturated alicyclic moietyhaving from 3-8 carbon atoms and includes, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term “cycloalkenyl” means an unsaturated alicyclicmoiety having from 3-8 carbon atoms and includes, for example,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyland cyclooctenyl. In the case of cycloalkenyl rings of from 5-8 carbonatoms, the ring may contain more than one double bond.

As used herein the term “aryl” refers to a mono-, bi- or tri-cycliccarbocyclic aromatic group, and to groups consisting of two covalentlylinked monocyclic carbocyclic aromatic groups. Illustrative of suchgroups are phenyl, biphenyl and napthyl.

As used herein the term “heteroaryl” refers to a 5- or 6-memberedaromatic ring containing one or more heteroatoms, and optionally fusedto a benzyl or pyridyl ring; and to groups consisting of two covalentlylinked 5- or 6-membered aromatic rings each containing one or moreheteroatoms; and to groups consisting of a monocyclic carbocyclicaromatic group covalently linked to a 5- or 6-membered aromatic ringscontaining one or more heteroatoms. Illustrative of such groups arethienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl,pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl,oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,4-([1,2,3]-thiadiazoly4-yl)phenyl and 5-isoxazol-3-ylthienyl.

As used herein the unqualified term “carbocyclyl” or “carbocyclic”refers to a 5-8 membered ring whose ring atoms are all carbon.

As used herein the unqualified term “heterocyclyl” or “heterocyclic”includes “heteroaryl” as defined above, and in particular means a 5-8membered aromatic or non-aromatic heterocyclic ring containing one ormore heteroatoms selected from S, N and O, and optionally fused to abenzene ring, including for example, pyrrolyl, furyl, thienyl,piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, thiazepinyl,pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl,piperazinyl, indolyl, benzimidazolyl, maleimido, succinimido,phthalimido and 1,3-dioxo-1,3-dihydro-isoindol-2-yl groups.

Unless otherwise specified in the context in which it occurs, the term“substituted” as applied to any moiety herein means substituted with upto four substituents, each of which independently may be (C₁-C₆)alkyl,benzyl, (C₁-C₆)alkoxy, phenoxy, hydroxy, mercapto, (C₁-C₆)alkylthio,amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl,nitro, -COOH, -CONH₂, -COR^(A), -COOR^(A), -NHCOR^(A), -CONHR^(A),-NHR^(A), -NR^(A)R^(B), or -CONR^(A)R^(B) wherein R^(A) and R^(B) areindependently a (C₁-C₆)alkyl group. In the case where “substituted”means benzyl, the phenyl ring thereof may itself be substituted with anyof the foregoing, except benzyl.

There are at least two actual or potential chiral centres in thecompounds according to the invention because of the presence ofasymmetric carbon atoms. The presence of several asymmetric carbon atomsgives rise to a number of diastereoisomers with R or S stereochemistryat each chiral centre. The invention includes all such diastereoisomersand mixtures thereof. Currently, the preferred stereoconfiguration ofthe carbon atom carrying the R₂ group is R; and that of the carbon atomcarrying the R₁ group (when asymmetric) is R.

In the compounds of formula (1) as defined above:

R₁ may be, for example, hydrogen, methyl, or trifuoromethyl. Hydrogen iscurrently preferred.

R₂ may be, for example:

optionally substituted C₁-C₈ alkyl, C₃-C₆ alkenyl, C₃-C₆ alkynyl orcycloalkyl;

phenyl(C₁-C₆ alkyld, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)-optionally substituted in the phenyl ring;

cycloalkyl(C₁-C₆ alkyl)-, cycloalkyl(C₃-C₆ alkenyl)- or cycloalkyl(C₃-C₆alkynyl)- optionally substituted in the cycloalkyl ring;

heterocyclyl(C₁-C₆ alkyl)-, heterocyclyl (C₃-C₆ alkenyl)- orheterocyclyl(C₃-C₆ alkynyl)- optionally substituted in the heterocyclylring; or

CH₃(CH₂)_(p)O(CH₂)_(q)- or CH₃(CH₂)_(p)S(CH₂)_(q)-, wherein p is 0, 1, 2or 3 and q is 1, 2or 3.

Thus, R₂ may be, for example,

C₁-C₆ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;

phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl (C₃-C₆alkynyl)-optionally substituted in the phenyl ring;

cycloalkyl(C₁-C₆ alkyl)-, cycloalkyl(C₃-C₆ alkenyl)- or cycloalkyl(C₃-C₆alkynyl)- optionally substituted in the phenyl ring;

heterocyclyl(C₁-C, alkyl)-, heterocyclyl(C₃-C₆ alkenyl)- orheterocyclyl(C₃-C₆ alkynyl)- optionally substituted in the heterocyclylring; or

4-phenylphenyl(C₁-C₆ alkyl)-, 4-phenylphenyl(C₃-C₆ alkenyl)-,4-phenylphenyl(C₃-C₆ alkynyl)-, 4-heteroarylphenyl(C₁-C₆ alkyl)-,4-heteroarylphenyl(C₃-C₆ alkenyl)-, 4-heteroarylphenyl(C₃-C₆ alkynyl)-,optionally substituted in the terminal phenyl or heteroaryl ring.

Specific examples of R₂ groups include methyl, ethyl, n- and iso-propyl,n- and iso-butyl, n-pentyl, iso-pentyl 3-methyl-but-1-yl, n-hexyl,n-heptyl, n-acetyl, n-octyl, methylsulfanylethyl, ethylsulfanylmethyl,2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl, 3-hydroxypropyl, allyl,3-phenylprop-3-en-1-yl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl,3-(2-chlorophenyl)prop-2-yn-1-yl, but-2-yn-1-yl, cyclopentyl,cyclohexyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl,cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, furan-2-ylmethyl,furan-3-methyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylmethyl,piperidinylmethyl, phenylpropyl, 4-chlorophenylpropyl,4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl,4-methylbenzyl, or 4-methoxybenzyl.

Presently preferred groups at R₂ are n-propyl, n-butyl, n-pentyl, benzyland cyclopentylmethyl.

R₃ and R₄, taken together with the carbon atoms to which they arerespectively attached, form an optionally substituted monocyclicsaturated carbocyclic or heterocyclic ring of 5 to 16 atoms, for examplefrom 5 to 8 atoms. Referring to formula (I), the carbon atom shown ascarrying the group R₃ is the one linking the ring to the rest of themolecule, and may arbitrarily be numbered as the 1- position in thering. The carbon atom shown as carrying the R₄ group is adjacent the1-position, and is oxo- (keto-) substituted. The remaining atoms in thering may all be carbons, or may include one or more hetero atoms —O—,—S— or —N(R₅)— wherein R₅ may be hydrogen, a C₁-C₄alkyl group, a phenylor benzyl group, or an acyl radical (for example phenylcarbonyl) orsulphonyl radical (for example 4-methoxyphenylsulphonyl or4-trifluoromethylphenylsulphonyl).

More than one hetero-atom may be present in the ring, but it will beappreciated that unstable combinations such as adjacent —O— atoms willnot be feasible. Where the ring contains an S atom, it may be oxidisedas a sulphinyl or sulphonyl. In one particular embodiment, the ring atomin the 3-position is —N(R₅)— wherein R₅ may be hydrogen or a C₁-C₄alkyl,phenyl or benzyl group. In another embodiment the ring contains one —O—or —S— atom. In still another embodiment the ring contains one —O— or—S— atom and a nitrogen atom separated by carbon atom(s). In yet anotherembodiment, the ring contains two or three nitrogen atoms.

In general, the radical formed by R₃ and R₄, taken together with thecarbon atoms to which they are respectively attached may be representedschematically as formula (II)

wherein rings A and B each have 5-8 ring atoms which may includeheteroatoms and which may each be substituted. Ring B is optional, andmay be a fused benzene ring or a second 5-8 membered saturatedcarbocyclic or heterocyclic ring.

The ring formed by R₃ and R₄, taken together with the carbon atoms towhich they are respectively attached, may be substituted (as definedabove). In one particular embodiment the ring atom in position n of ann-membered ring (n being 5, 6, 7 or 8 and numbering from position 1,with the oxo-substituted carbon atom being position 2) may be a carbonatom substituted by one or two methyl groups, and the ring atom inposition n-1 may be a sulphur atom. Examples of such rings, formed by R₃and R₄, taken together with the carbon atoms to which they arerespectively attached, are those of the following structure (III):

wherein R₈ may be hydrogen or a C₁-C₄alkyl (eg methyl, ethyl orn-propyl), cycloalkyl (eg cyclopentyl) phenyl or benzyl group, and R,may be hydrogen or a C₁-C₄alkyl (eg isobutyl), phenyl or benzyl group,or R₈ and R₉ taken together represent a divalent —(CH₂)_(p)— radicalwherein p is 3 or 4.

Specific examples of radicals having the general structure (II) include

4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6-yl

3-benzyl4,7,7- trimethyl-5-oxo-[1,4]thiazepan-6-yl

3,3-dimethyl-5-oxo- hexahydro-pyrrolo[2,1-c][1,4]thiazepin4-yl

7,7-dimethyl-5-oxo- octahydro-8-thia-4a-aza-benzocyclohepten-6-yl

7,7-dimethyl-5-oxo-octahydro-8-thia4a-aza-benzocycloocten-6-yl

4-ethyl-7,7-dimethyl-5- oxo-[1,4]thiazepan-6-yl

7,7-dimethyl-5-oxo4-propyl-[1,4]thiazepan-6-yl

7,7-dimethyl-5-oxo4-phenyl-[1,4]thiazepan-6-yl

7,7-dimethyl-5-oxo-4- phenyl-[1,4]thiazepan-6-yl

1-methyl-2-oxo-azepan-3-yl

1-benzyl-2-oxo-piperidin-3-yl

1-methyl-2,5-dioxo-pyrrolidin-3-yl

4-methyl-3,5-dioxo-[1,4]oxazepan-6-yl

4-methyl-3,5-dioxo-[1,4]thiazepan-6-yl

1-methyl-2,7-dioxo-azepan-3-yl

1-methyl-2,6-dioxo-piperidin-3-yl

1-methyl-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl

2-methyl-3-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin4-yl

1-methyl-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl

9-methyl-8-oxo-6,7,8,9-tetrahydro-5-oxa-9-aza-benzocyclohepten-7-yl

1-methyl-7-oxo-[1,4]diazepan-6-yl

1-(4-methoxy-benzenesulfonyl)4-methyl-5-oxo-[1,4]diazepan-6-yl

1-benzoyl-4-methyl-5-oxo-[1,4]diazepan-6-yl

1,4-dimethyl-5-oxo-[1,4]diazepan-6-yl

3-benzyl-5-oxo-[1,4]thiazepan-6-yl

3-benzyl-4-methyl-1,1,5-trioxo-[1,4]thiazepan-6-yl

2-benzyl-7-oxo-[1,4]oxathiepan-6-yl and

4-oxo-thiepan-3-yl.

Specific examples of compounds of the invention include thosespecifically named and characterised in the Examples herein.

Compounds of the invention may be prepared by a process comprisingcoupling an acid of formula (IV) with an amine of formula (V)

wherein R₁, R₂, R₃ and R₄ are as defined in relation to formula (I). TheN-hydroxy group of compound (IV) is preferably protected during thecoupling reaction, and the hydroxy group subsequently regenerated.

Compounds of formula (V) may be prepared by cyclisation of compounds offormula (VA)

wherein one of R₁₃ and R₁₄ contains a reactive group capable of reactingwith a reactive partner site on the other of R₁₃ and R₁₄ to form acovalent bond between R₁₃ and R₁₄, thereby forming the desired ring,represented in formula (1) by R₃ and R₄ taken together with the carbonatoms to which they are respectively attached. During this cyclisationreaction, the amino group of (VA) will usually be protected, and theamino group subsequently released. Such intramolecular cyclisations, forexample intramolecular condensation reactions, are known in thesynthetic chemistry art. Alternatively, compounds of formula (V) may beprepared by reaction of a compound of formula (VB)

with a bifunctional linker schematically represented as X₁—L—X₂, whereinX₁ is a reactive group capable of reacting with a reactive partner sitein R₂₃, X₂ is a reactive group capable of reacting with a reactivepartner site in R₂₄, and R₂₃, R₂₄ and L are chosen such that afterreaction of X₁ and X₂ with their respective reactive partner sites, Lforms a covalently linked bridge between R₂₃ and R₂₄ and completes thedesired ring, represented in formula (I) by R₃ and R₄ taken togetherwith the carbon atoms to which they are respectively attached. Again,during this cyclisation reaction, the amino group of (VA) will usuallybe protected, and the amino group subsequently released.

Again such intra molecular cyclisation by ring formation is known in theart of synthetic chemistry. Example 1 below is one illustration of sucha reaction. Such reactions are preferably carried out at high dilutionto maximise intramolecular rather than intermolecular bridge formation.

Compounds of the invention may also be accessible by cyclisation of acompound of formula (VI)

wherein R₁ and R₂ are as defined in relation to formula (1), and R₁₃ andR₁₄ are as defined in relation to formula (VA). Preferably the N-formylhydroxylamine group in (VI) is protected during the cyclisation reactionand the protecting group subsequently removed, or may be attached to asolid phase support (eg a resin), and subsequently released.

Alternatively, compounds of the invention may be accessible by reactionof a compound of formula (VIA)

with a bifunctional linker schematically represented as X₁—L—X₂, whereinR₁ and R₂ are as defined in relation to formula (1), and X₁, X₂ and Lare as defined in relation to formula (VB). Preferably the N-formylhydroxylamine group in (V) is protected during the bridging reaction andthe protecting group subsequently removed, or may be attached to a solidphase support (eg a resin), and subsequently released.

Antibacterial compositions with which the invention is concerned may beprepared for administration by any route consistent with thepharmacokinetic properties of the active ingredient(s).

Orally administrable compositions may be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions.Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricant, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants for example potato starch, or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavouring or colouring agents.

For topical application to the skin, the active ingredient(s) may bemade up into a cream, lotion or ointment. Cream or ointment formulationswhich may be used for the drug are conventional formulations well knownin the art, for example as described in standard textbooks ofpharmaceutics such as the British Pharmacopoeia.

The active ingredient(s) may also be administered parenterally in asterile medium. Depending on the vehicle and concentration used, thedrug can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as a local anaesthetic, preservative andbuffering agents can be dissolved in the vehicle. Intra-venous infusionis another route of administration for the compounds used in accordancewith the invention.

Safe and effective dosages for different classes of patient and fordifferent disease states will be determined by clinical trial as isrequired in the art. It will be understood that the specific dose levelfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

The finding that compounds with PDF inhibitory activity can inhibit orprevent bacterial growth, opens up a novel approach for identifying newantibacterial agents by screening test compounds for activity asinhibitors of PDF in vitro, followed by confirmation of theirantibacterial ability using bacterial growth inhibition studies. Thisfinding also makes available (i) the use of compounds with PDFinhibitory activity as antibacterial agents, and (ii) a method for thetreatment of bacterial infection or contamination by applying oradministering a compound which inhibits the activity of bacterial PDF.

THE FOLLOWING EXAMPLE ILLUSTRATES AN EMBODIMENT OF THE INVENTION.

The following abbreviations have been used throughout:

DMF N,N-Dimethylformamide

EDC N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride

TFA Trifluoroacetic acid

HOAt 1-Hydroxy-7-aza-benzotriazole

HOBt 1-Hydroxybenzotriazole

HPLC High performance liquid chromatography

LRMS Low resolution mass spectrometry

TLC Thin layer chromatography

¹H and ¹³C NMR spectra were recorded using a Bruker AC 250E spectrometerat 250.1 and 62.9 MHz, respectively. Mass spectra were obtained using aPerkin Elmer Sciex API 165 spectrometer using both positive and negativeion modes.

The starting materials 2R-(tert-butoxyamino-methyl)-hexanoic acid and2R-[(tert-butoxy-formyl-amino)-methyl]-hexanoic acid pentafluorophenylester were prepared as described below (see also Scheme 1). The startingmaterials 2R-[(benzyloxy amino)-methyl]-hexanoic acid and2R-[(benzyloxy-formyl-amino)-methyl]-hexanoic acid pentafluorophenylester were prepared in a similar fashion.

STEP 1: 2-Butyl-acrylic Acid

Butylmalonic acid (25 g, 156 mmol) was dissolved in ethanol (250 ml) and37% formaldehyde solution (15.45 ml, 156 mmol) was added followed bypiperidine (47 ml, 624 mmol). The mixture was stirred overnight at 80°C. under a reflux condenser. The solvents were removed under reducedpressure and the residue was diluted with 1M hydrochloric acid andextracted with dichloromethane (3×30 ml). The combined organic extractswere washed with brine, dried over anhydrous magnesium sulfate, filteredand evaporated to afford the desired product as a yellow oil (25 g, withresidual solvent). ¹H-NMR: δ (CDCl₃), 10.04 (1H, br s), 6.22 (1H, s),5.57 (1H, d, J=1.3 Hz), 2.30 (2H, t, J=6.9 Hz), 1.38 (4H, m), and 0.91(3H, t, J=7.2 Hz).

STEP 2: 4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one

2-Butyl acrylic acid (21.5 g, 168 mmol) was dissolved in dry THF (500ml) and cooled to −78° C. under a blanket of argon. Triethylamine (30ml, 218 mmol) and pivaloyl chloride (21 ml, 168 mmol) were added at sucha rate that the temperature remained below −60° C. The mixture wasstirred at −78° C. for 30 minutes, warmed to room temperature for 2hours and finally cooled back to −78° C.

In a separate flask, 4S-benzyl-5,5-dimethyl-oxazolidin-2-one wasdissolved in dry THF (500 ml) and cooled to −78° C. under a blanket ofargon. n-Butyllithium (2.4 M solution in hexanes, 83 ml, 200 mmol) wasadded slowly and the mixture was stirred for 30 minutes at roomtemperature. The resulting anion was transferred via a cannula into theoriginal reaction vessel. The mixture was allowed to warm to roomtemperature and was stirred overnight at room temperature. The reactionwas quenched with I M potassium hydrogen carbonate (200 ml) and thesolvents were removed under reduced pressure. The residue waspartitioned between ethyl acetate and water. The organic layer waswashed with brine, dried over anhydrous magnesium sulphate, filtered andconcentrated under reduced pressure to give an orange oil. TLC analysisrevealed the presence of unreacted chiral auxiliary in. addition to therequired product. A portion of the material (30 g) was dissolved indichloromethane and flushed though a silica pad to give pure titlecompound as a yellow oil (25.3 g). ¹H-NMR; δ (CDCl₃), 7.31-7.19 (5H, m),5.41 (2H, s), 4.51 (1H, dd, J=9.7, 4.2 Hz), 3.32 (1H, dd, J=14.2, 4.2Hz), 2.82 (1H, dd, J=14.2, 9.7 Hz), 2.40-2.34 (2H, m), 1.48-1.32 (4H,m), 1.43 (3H, s), 1.27 (3H, s) and 0.91 (3H, t, J=7.1 Hz). Some chiralauxiliary was recovered by flushing the silica pad with methanol.

STEP 3:4S-tert-Butyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one-2R-(tert-butoxyamino-methyl)-hexanamide

4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one (5 g, 15.92mmol) was dissolved in ethanol (20 ml). O-tert-butylhydroxylaminehydrochloride (2.4 g, 19.10 mmol) and triethylamine (2.75 ml, 19.90mmol) were then added and the reaction mixture stirred at 30° C. for 24hours. Solvent was removed under reduced pressure and the residuedissolved in ethyl acetate and washed successively with 1 M hydrochloricacid, saturated sodium hydrogen carbonate and brine, dried overmagnesium sulphate and filtered. Solvents were removed under reducedpressure to provide the title compound (5.62 g, 13.89 mmol, 87%) as acolourless oil. ¹H-NMR; δ (CDCl₃), 7.29 (5H, m), 5.07 (1H, s), 4.51 (1H,dd, J=3.2, 10.0 Hz), 4.01 (1H, m), 3.25 (1H, dd, J=2.9,14.4 Hz), 3.03(2H, m), 2.84 (1H, dd, J=4.3, 9.8 Hz), 1.69 (1H, m), 1.49 (2H, m), 1.34(6H, s), 1.25 (3H, m), 1.13 (9H, s) and 0.87 (3H, t, J=6.9 Hz). LRMS:+veion 405 [M+H].

STEP 4: 2R-(tert-Butoxyamino-methyl)-hexanoic Acid

4S-tert-Butyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one-2R-tert-butoxyamino-methyl)-hexanamide(5.62 g, 13.89 mmol) was dissolved in THF (40 ml) and water (10 ml) andcooled to 0° C. and treated with lithium hydroxide (0.64 g, 15.28 mmol).The solution was stirred for 30 minutes at 0° C., then overnight at roomtemperature. The reaction was acidified to pH4 with 1 M citric acid andthe solvents were removed. The residue was partitioned betweendichloromethane and 1 M sodium carbonate. The basic aqueous layer wasacidified to pH4 with 1M citric acid and extracted three times withethyl acetate. The combined organic layers were dried over anhydrousmagnesium sulfate, filtered and concentrated to provide the titlecompound as a colourless oil (2.0 g, 66%). ¹H-NMR; δ (CDCl₃), 7.31 (2H,br s), 3.14 (1H, dd, J=9.1, 12.7 Hz), 2.99 (1H, dd, J=4.2, 12.7 Hz),2.63 (1H, m), 1.65 (1H, m), 1.54 (1H, m), 1.29 (4H, m), 1.17 (9H, s) and0.90 (3H, t, J=7.2 Hz).

STEP 5: 2R-[(tert-Butoxy-formyl-amino)-methyl]-hexanoic Acid

To a solution of 2R-(tert-butoxyamino-methyl)-hexanoic acid (4 g, 18.5mmol) in dichloromethane (40 ml) at 0° C. was added formic aceticanhydride (4.1 ml, 46.25 mmol) and triethylamine (2.4 ml, 18.5 mmol).After 2 hours the solution was washed with water, saturated sodiumhydrogen carbonate and brine, dried over anhydrous magnesium sulphateand filtered. The filtrate was evaporated to provide the title compoundas an oil, which was taken on to Step 6 without further purification.LRMS: +ve ion 245 (M+H).

STEP 6: 2R-[(tert-Butoxy-formyl-amino)-methyl]-hexanoic AcidPentafluorophenyl Ester

2R-[(tert-butoxy-formyl-amino)-methyl]-hexanoic acid (1.73 g, 7.06 mmol)was dissolved in dichloromethane (60 ml) and cooled to 0° C.Pentafluorophenol (1.43 g, 7.77 mmol) and EDC (1.5 g, 7.77 mmol) wereadded and the resulting solution stirred at 0° C. for 0.5 hours and roomtemperature for 4 hours. The solution was then washed with 1M sodiumcarbonate and brine, dried over magnesium sulphate and filtered.Solvents were removed under reduced pressure to provide the titlecompound as a colourless oil (2.6 g, 6.33 mmol, 90%. LRMS: +ve ion 412(M+H).

EXAMPLE 1 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl) amide

The title compound was prepared by the method described below (see alsoScheme 2):

STEP A: 2R-tert-Butoxycarbonylamino-3-mercapto-3-methyl-butyric Acid

L-Penicillamine (15 g, 0.1 mol) was suspended in methanol (250 ml) andcooled to 0° C. Triethylamine (14 ml, 0.1 mol) and di-tert-butyldicarbonate (24 g, 0.11 mol) in methanol (25 ml) were then addedsequentially. The reaction mixture was allowed to stir at roomtemperature overnight, after which the solvents were removed underreduced pressure. The residue was dissolved in ethyl acetate (100 ml)and washed with 1N hydrochloric acid (100 ml), water (50 ml) and brine(50 ml), dried over anhydrous magnesium sulphate, filtered andevaporated to provide the title compound as a white solid. (28.1 g,including residual solvent). ¹H-NMR; δ (CDCl₃), 9.13 (1H, brs), 5.52(1H, d, J=9.9Hz), 4.34 (1H, d, J =9.3 Hz), 2.01 (1H, s), 1.53 (3H, s),1.46 (9H, s) and 1.42 (3H, s).

STEP B: {1R-[(2-Hydroxy-ethyl)-methyl-carbamoyl]-2-mercapto-2-methyl-propyl}-carbamicAcid tert-butyl Ester

2R-tert-Butoxycarbonylamino-3-mercapto-3-methyl-butyric acid (5 g, 20mmol) was dissolved in DMF (30 ml) and the solution was cooled to 0° C.EDC (4.6 g, 24 mmol) and HOBt (3.25 g, 24 mmol) were added and themixture was stirred for 15 minutes. N-(2-Hydroxy-ethyl)-N-methyl amine(1.16 ml, 20 mmol) was added and the reaction was allowed to warm toroom temperature and was stirred overnight. The solvent was removedunder reduced pressure and the residue was dissolved in dichloromethane,washed successively with 1 M hydrochloric acid, saturated sodiumhydrogen carbonate and brine, dried over magnesium sulphate andfiltered. Solvents were removed under reduced pressure and the residuewas purified by flash column chromatography (silica gel, 5% methanol indichloromethane) to provide the title compound as a colourless oil (3.58g, 58%). ¹H-NMR; δ (CDCl₃), 5.67 (1H, d, J=9.5 Hz), 5.55 (1H, d, J=8.7Hz), 4.68 (2H, dd, J=9.7, 13.8 Hz), 3.79 (2H, m), 3.27 (3H, s), 2.77 (1H, br s), 1.81 (1H, s), 1.44 (9H, s), 1.45 (3H, s) and 1.43 (3H, s).

STEP C: (4,7,7-Trimethyl-5-oxo-[1,4]thiazepan-6-yl)-carbamic Acidtert-Butyl Ester

A solution of{1R-[(2-Hydroxy-ethyl)-methyl-carbamoyl]-2-mercapto-2-methyl-propyl}-carbamicacid tert-butyl ester (3 g, 9.8 mmol) in THF (100 ml) was treated withtriphenylphosphine (3.28 g, 12.5 mmol) and diisopropylazodicarboxylate(2.45 ml, 12.5 mmol) and the resulting solution stirred at roomtemperature for 6 hours. Solvent was removed under reduced pressure andthe residue purified by flash column chromatography (Biotage, 40% ethylacetate in hexanes) to provide the title compound as a colourless oil(1.0 g, 35%). ¹H-NMR; δ (CDCl₃), 5.83 (1H, d, J=7.8 Hz), 4.89 (1H, d,J=9.4 Hz), 4.00 (1H, dd, J=3.6, 9.6 Hz), 3.56 (1H, dd, J=5.4, 9.8 Hz),3.04 (3H, s), 2.69 (1H, ddd, J=3.5, 5.5, 11.0 Hz), 1.44 (9H, s), 1.36(3H, s) and 1.19 (3H, s). LRMS: +ve ion 289 [M+H].

STEP D: 6S-Amino4,7,7-trimethyl-[1,4]thiazepan-5-one

A solution of (4,7,7-trimethyl-5-oxo-[1,4thiazepan-6-yl)carbamic acidtert-butyl ester (1 g, 3.47 mmol) in dichloromethane (10 ml) and TFA (10ml) was allowed to stand at 0° C. overnight. Solvents were removed underreduced pressure and the residue was dissolved in methanol (30 ml) andwater (3 ml) and treated with Dowex400 resin to pH 8. The resin wasremoved by filtration and washed well with methanol. The filtrate andwashings were evaporated to provide the title compound as a yellow oil(0.65 g, quant.). ¹H-NMR; δ (CD₃OD), 3.94 (1H, s), 3.89 (1H, t, J=6.8Hz), 3.62 (1H, dt, J=5.3, 15.5 Hz), 3.00 (3H, s), 2.82 (2H, t, J=5.3Hz), 1.38 (3H, s) and 1.16 (3H, s). LRMS: +ve ion 189 [M+H].

STEP E: 2R-[(tert-Butoxy-amino)-methyl]-hexanoicAcid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl)amide

2R-(tert-Butoxy-amino-methyl)-hexanoic acid (0.4 g, 1.85 mmol) wasdissolved in DMF (30 ml) and the solution was cooled to 0° C. EDC (0.42,2.18 mmol) and HOAt (25 mg, 0.185 mmol) were added and the mixture wasstirred for 15 minutes. 6S-Amino4,7,7-trimethyl-[1,4]thiazepan-5-one(0.5 g, 1.85 mmol) was added and the reaction was allowed to warm toroom temperature and was stirred overnight. The solvent was removedunder reduced pressure and the residue was dissolved in dichloromethane,washed successively with 1 M hydrochloric acid, saturated sodiumhydrogen carbonate and brine, dried over magnesium sulphate andfiltered. Solvents were removed under reduced pressure and the residuewas purified by flash column chromatography (Biotage, 50% ethyl acetatein hexanes) to provide the title compound as a colourless oil (160 mg,22%). ¹H-NMR; δ (CDCl₃), 6.99 (1H, d, J=8.2 Hz), 5.25 (1H, d, J=8.3 Hz),5.00 (1H, s), 4.06 (1H, ddd, J=4.9, 10.5, 16.5 Hz), 3.53 (1H, dt, J=4.6,16.8 Hz), 3.07 (1H,dd, J=7.1, 12.5 Hz), 3.04 (3H, s), 2.94 (1H,dd,J=4.6, 8.5 Hz), 2.84 (1H, dd, J=4.5, 9.5 Hz), 2.73 (1H, dt, J=3.9, 14.0Hz), 1.55 (1H, m), 1.37 (3H, s), 1.28 (5H, m), 1.22 (3H, s), 1.16 (9H,s) and 0.87 (3H, t, J=7.7 Hz). LRMS: +ve ion 387 [M+H].

STEP F: 2R[(tertButoxy-formyl-amino)-methyl]-hexanoicAcid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl) amide

A solution of 2R[(tertButoxy-amino)-methyl]-hexanoic acid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl) amide (1 60 mg, 0.41 mmol)in dichloromethane (5 ml) was treated with formic acetic anhydride (0.2ml) and stirred for 5 hours. Solvents were removed under reducedpressure to provide the title compound as a colourless oil (180 mg,97%). ¹H-NMR; δ (CDCl₃, rotamers), 8.3 (1H, s), 7.17 (0.33H, d, J=7.4Hz), 6.92 (0.66H, d, J=7.8 Hz), 5.31 (0.33H, d, J=9.4 Hz), 5.19 (0.66H,d, J=8.3 Hz), 4.03 (1H, m), 3.90 (1H, m), 3.68 (1H, m), 3.52 (1H, dt,J=5.7, 16.6 Hz), 3.03 (3H, s), 2.78 (3H, m), 1.56 (1H, m), 1.41 (2H, m),1.35 (3H, s), 1.27 (9H, s), 1.23 (3H, m), 1.20 (3H, s) and 0.86 (3H, t,J=7.6 Hz). LRMS: +ve ion 416 [M+H].

STEP G: 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoicAcid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl) amide

2R-[(tert-Butoxy-formyl-amino)-methyl]-hexanoic acid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl) amide (180 mg, 0.4 mmol)was dissolved in TFA (10 ml) and stirred at room temperature for 6hours. Solvent was removed under reduced pressure, azeotroping severaltimes with toluene. The title compound was purified using preparativeHPLC (methanol:water, gradient method) as a white solid (80 mg, 0.22mmol, 54%). ¹H-NMR; δ (CDCl₃, (rotamers)), 8.39 (0.5H, s), 7.84 (0.5H,s), 7.19 (0.5H, d, J=8.3 Hz), 6.99 (0.5H, d, J=8.3 Hz), 5.28 (0.5H, d,J=8.2 Hz), 5.23 (0.5H, d, J=8.2 Hz), 4.05 (1.5H, m), 3.86 (0.5H, dd,J=9.9, 14.1 Hz), 3.52 (2H, m), 3.06 (1.5H, s), 3.04 (1.5H, s), 2.85(1.5H, m), 2.74 (1.5H, m), 1.61 (1H, m), 1.43 (1H, m), 1.32 (1H, s),1.30 (4H, m), 1.26 (2H, s), 1.21 (1H, s), 1.17 (2H, s), and 0.88 (3H, t,J=6.7 Hz). ¹³C-NMR; δ (CDCl₃, (rotamers)), 171.1, 59.4, 53.1, 52.8,51.5, 48.4, 46.6, 45.2, 44.0, 36.7, 36.5, 30.3, 29.6, 27.5, 27.2, 24.0,23.0 and 14.2. LRMS: +ve ion 360 [M+H], 382 [M+Na], −ve ion 358 [M−H].

EXAMPLE 2 2-[(Formyl-hydroxy-amino)-methyl]-hexanoicAcid(5,5-Dimethyl-7-oxo-[1,4]oxathiepan-6-yl)-amide

STEP A:2S-tert-Butoxycarbonylamino-3-(2-hydroxy-ethylsulfanyl)-3-methyl-butyricAcid

2S-tert-Butoxycarbonylamino-3-mercapto-3-methyl-butyric acid (1 g, 4mmol) was dissolved in a 1:1 mixture of ethanol and 1M sodium hydroxidesolution (20 ml). 2-Bromoethanol (0.34 ml, 4.8 mmol) was then added andthe resulting solution heated at 40° C. overnight. Ethanol was removedin vacuo and the residue diluted with water (10 ml), acidified to pH 1with 1M hydrochloric acid and extracted with dichloromethane (3×10 ml).The combined organic layer was combined, washed with brine (10 ml),dried over anhydrous magnesium sulphate, filtered and evaporated toprovide the title compound as a white gum (1.1 g, 3.75 mmol, 94%).¹H-NMR; δ (CDCl₃); 5.57 (1H, d, J =9.1 Hz), 5.38 (1H, br s), 4.40 (1H,d, J=9.2 Hz), 3.76 (2H, m), 2.83 (2H, t, J=5.8 Hz), 1.45 (9H, s), 1.42(3H, s) and 1.35 (3H, s). LRMS: +ve ion 294 [M+H], 316 [M+Na], −ve ion292 (M−H).

STEP B: (5,5-Dimethyl-7-oxo-[1,4]oxathiepan-6S-yl)-carbamic Acidtert-Butyl Ester

A solution of2S-tert-butoxycarbonylamino-3-(2-hydroxy-ethylsulfanyl)-3-methyl-butyricacid (1.1 g, 3.75 mmol) and ethyl chloroformate (0.4 ml, 4.12 mmol) indichloromethane was cooled to 0° C. and treated dropwise withtriethylamine (0.57 ml, 4.12 mmol) and dimethylaminopyridine (46 mg,0.375 mmol). The resulting solution was stirred at 0° C. for 0.5 hoursthen diluted with dichloromethane (15 ml), washed successively with 1 Mhydrochloric acid, saturated sodium hydrogen carbonate and brine, driedover magnesium sulphate and filtered. Solvents were removed in vacuo toprovide the title compound (0.86 g, 3.12 mmol, 83%) as a colourless oil.¹H-NMR; δ (CDCl₃); 5.59 (1H, d, J=8.1 Hz), 5.03 (1H, d, J=8.6 Hz), 4.65(2H, m), 3.05 (1H, ddd, J=3.7, 8.4, 15.7 Hz), 2.79 (1H, ddd, J=2.3, 4.8,15.8 Hz), 1.45 (9H, s), 1.39 (3H, s) and 1.27 (3H, s) and 2.27 (3H, s).LRMS: +ve ion 276 [M+H], 298 [M+Na].

STEP C: 6S-Amino-5,5-dimethyl-[1,4]oxathiepan-7-one

A solution of (5,5-dimethyl-7-oxo-[1,4]oxathiepan-6S-yl)-carbamic acidtert-butyl ester (0.86 g, 3.12 mmol) in 5% aqueous trifluoroacetic acid(20 ml) was allowed to stand at 0° C. for 16 hours. Solvents wereremoved in vacuo, azeotroping with toluene, then the residue dissolvedin 10% aqueous methanol (30 ml) and adjusted to pH 8 with Dowex 1×8 400resin. This was filtered and concentrated under reduced pressure toprovide the title compound as a colourless gum (0.72 g, 4 mmol, 100%).¹H-NMR; δ (CDCl₃); 4.71 (1H, ddd, J=2.7, 5.5, 12.9 Hz), 4.54 (1H, ddd,J=2.0, 8.9, 14.9 Hz), 4.33 (1H, s 3.02 (1H, ddd, J=2.8, 8.9, 15.9 Hz),2.81 (1H, ddd, J=1.9, 5.7, 11.6 Hz), 2.33 (2H, s), 1.46 (3H, s) and 1.34(3H, s).

STEP D: 2-[(Formyl-hydroxy-amino)-methyl]-hexanoicAcid(5,5-Dimethyl-7-oxo-[1,4]oxathiepan-6-yl)-amide

A solution of 6S-amino-5,5-dimethyl-[1,4]oxathiepan-7-one (320 mg, 1.83mmol) and 2R-[(tert-butoxy-formyl-amino)-methyl]-hexanoic acidpentafluorophenyl ester (0.5 g, 1.22 mmol) in DMF (5 ml) was stirred atroom temperature for 5 days. DMF was removed in vacuo and the residuedissolved in dichloromethane (30 ml), washed successively with 1 Mhydrochloric acid, saturated sodium hydrogen carbonate and brine, driedover magnesium sulphate and filtered. Solvents were removed underreduced pressure and the residue dissolved trifluoroacetic acid (10 ml)and stirred at room temperature for 6 hours. Solvent was removed invacuo, azeotroping with toluene, and the title compound was purifiedusing preparative HPLC (methanol:water, gradient method) (80 mg, 0.22mmol, 54%) as a white solid. ¹H-NMR; δ (CDCl₃, (rotamers)), 8.36 (0.33H,s), 7.85 (0.66H, s), 7.18 (0.66H, d, J=7.3 Hz), 7.10 (0.33H, d, J=7.9Hz), 5.41 (1H, d, J=8.5 Hz), 4.69 (2H, m), 3.75 (2H, m), 3.42 (1H, dd,J=4.0, 14.2 Hz), 3.09 (1H, m), 2.94 (1H, m), 2.80 (2H, m), 1.65 (1H, m),1.35 (3H, s), 1.32 (5H, m), 1.30 (3H, s), and 0.89 (3H, t, J=6.5 Hz).¹³C-NMR; δ (CDCl₃ (rotamers)), 173. 8, 172.8, 71.6, 71.3, 61.2, 60.9,52.3, 49.1, 46.0, 44.7, 43.4, 43.2, 30.4, 30.2, 29.8, 28.8, 27.5, 26.5,23.0 and 14.3. LRMS: +ve ion 347 (M+H), 369 (M+Na), −ve ion 345 (M−H).

The compounds of Examples 3 to 5 were prepared by parallel synthesis insolution from 2R-tert-butyloxyamino-methyl)-hexanoic acidpentafluorophenyl ester and penicillamine derived [1,4]oxathiepanamines. The requisite amines were prepared as outlined in Scheme 3 andas described in detail below.

STEP A: (2,2-Dimethyl4-oxo-thietan-3-yl)-carbamic Acid tert-Butyl Ester

2S-tert-Butoxycarbonylamino-3-mercapto-3-methyl-butyric acid (8 g, 32.10mmol) was dissolved in dichloromethane (300 ml) and cooled to 0° C.Pentafluorophenol (6.5 g, 35.31 mmol) and EDC (7.4 g, 38.52 mmol) werethen added and the resulting solution allowed to warm to roomtemperature and stirred for 2 hours, washed successively with 1 Mhydrochloric acid, 1M sodium carbonate and brine, dried over magnesiumsulphate and filtered. Solvents were removed in vacuo to provide thetitle compound as a white solid (7.45 g, 100%). ¹H-NMR; δ (CDCl₃); 5.41(1H, d, J=8.4 Hz), 5.30 (1H, br s), 1.83 (3H, s), 1.64 (3H, s) and 1.45(9H, s).

STEPS B-D:

(2,2-Dimethyl4-oxo-thietan-3-yl)-carbamic acid tert-butyl ester (0.62 g,2.7 mmol) and the required 2-hydroxy amine (2-hydroxymethylpiperidinefor Example 3, N-ethyl-ethanolamine for Example 4 orN-propyl-ethanolamine for Example 5; 2.7 mmol) were dissolved in DMF (5ml) and stirred at ambient temperature over night then concentrated invacuo. The residue was dissolved in tetrahydrofuran (20 ml) andtri-n-butyl phosphine (1 ml, 4.05 mmol) added and the solution cooled to0° C. 1,1′-(Azodicarbonyl)dipiperidine (1 g, 4.05 mmol) was the addedand the solution stirred at 0° C. for 5 minutes and at ambienttemperature for 4 hours, then diluted with hexane (5 ml), filteredthrough a cartridge containing a plug of silica and concentrated invacuo. The residue was dissolved in 5% aqueous TFA (20 ml) and allowedto stand at 0° C. overnight then concentrated in vacuo, azeotroping withtoluene. The residue was dissolved in dichloromethane (20 ml) andextracted into water (3×10 ml). The aqueous solution was adjusted to pH8 with 1M sodium carbonate and extracted with dichloromethane, driedover magnesium sulphate and filtered. Solvents were removed in vacuo toprovide the required amines. A solution of the amine (1.1 equivalent)and 2R-tert-butyloxyamino-methyl)-hexanoic acid pentafluorophenol ester(1 equivalent) in dichloromethane (5 ml) was stirred at ambienttemperature overnight.

The solution was then treated with carbonate resin (3 mmol/g, 3equivalents) for 1 hour, filtered and treated with sulphonic acid resin(1 mmol/g, 3 equivalents) for a further 1 hour. The suspension wasfiltered and concentrated to dryness in vacuo. The residue was dissolvedin trifluoroacetic acid (10 ml) and stirred at ambient temperature for 6hours then concentrated in vacuo. Final compounds were purified usingpreparative HPLC (methanol: water, gradient method).

EXAMPLE 3 2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(7,7-dimethyl-5-oxo-octahydro-8-thia-4a-aza-benzocyclohepten-6-yl)-amide

Yellow oil, 4.6 mg. LRMS +ve ion: 400 (M+H).

EXAMPLE 4 2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4-ethyl-7,7-dimethyl-5-oxo-[1,4]thiazapan-6-yl)-amide

Colourless oil, 24 mg. LRMS +ve ion: 374 (M+H).

EXAMPLE 5 2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4-propyl-7,7-dimethyl-5-oxo-[1,4]thiazapan-6-yl)-amide

Yellow oil, 40 mg. LRMS +ve ion: 388 (M+H), −ve ion: 386 (M−H).

EXAMPLE 6 2R-[(Formyl-hydroxy-amino)methyl]-hexanoic acid(1benzyl-2-oxo-piperidin-3S-yl) amide

Step A: 5-Benzylamino-2S-benzyioxycarbonylamino-pentanoic Acid

Benzaidehyde (0.42 ml, 4.25 mmol) was added to a stirred suspension of5-amio-2S-benzyloxycarbonylamino-pentanoic acid (1.0 g, 3.75 mmol),triethylamine (0.62 ml, 4.5 mmol) and magnesium sulfate (0.96 g, 8.1mmol) in methanol (10 ml) and the stirring continued at room temperatureovernight. The reaction mixture was then filtered and concentrated invacuo. The residue was dissolved in methanol, cooled in an ice bath andsodium cyanoborohydride (0.28 g, 7.5 mmol) was added portion-wise. After2 hours, acetone (30 ml) was added to quench the reaction and thereaction mixture was concentrated in vacuo to leave the crude titlecompound as brown solid (1.6 g) which was taken on to the next stepwithout further purification.

Step B: (1-Benzyl-2-oxo-piperidin-3S-yl)-carbamic Acid Benzyl Ester

5-Benzylamino-2S-benzyloxycarbonylamino-pentanoic acid was diluted DMF(16 ml) and cooled in an ice bath. HOBt (51 mg, 0.37 mmol) and EDC (0.75g, 3.9 mmol) were added to the stirred solution and the stirringcontinued for 60 h. The reaction mixture was concentrated in vacuo andthe residue was dissolved in ethyl acetate. The organic extract waswashed with 1M hydrochloric acid (15 ml), 1M sodium carbonate (15 ml),brine (15 ml), dried over magnesium sulphate and concentrated underreduced pressure. Silica column chromatography (dichloromethane:methanol 98:2 to 80:20) gave the pure title compound (0.3 g, 25%).¹H-NMR; δ (CDCl₃) 7.30 (10H, m), 5.85 (1H, s), 5.12 (2H, s), 4.64 (2H,m), 4.15 (1H, m), 3.24 (2H, m), 1.88-1.52 (4H, m). LRMS: +ve ion 339[M+H].

Step C: 3S-Amino-1Benzyl-piperidin-2-one

Palladium on charcoal (10%) was added to a stirred solution of(1-benzyl-2-oxo-piperidin-3S-yl)-carbamic acid benzyl ester (0.29 g,0.86 mmol) in ethanol (40 ml) under argon. H₂ was bubbled through thesuspension for 2 h. The mixture was purged with argon before thecatalyst was removed by filtration. The solvent was removed in vacuo toafford the title compound as a colourless oil (0.17 g, 98%).¹H-NMR: δ(CDCl₃) 7.26 (5H, m), 4.49 (2H, m) 3.52 (1H, m), 3.23 (2H, m), 2.26-1.73(4H, m). LMRS: +ve ion 205 [M+H]

Step D: 2R-[(Benzyloxy-amino-formyl)-methyl]-hexanoicAcid(1-benzyl-2-oxo-piperidin-3S-yl) amide

To a solution of 2R-[(benzyloxy-formyl-amino)-methyl]-hexylpentafluorophenyl ester (155 mg, 0.81 mmol) in dichloromethane (4 ml)was added 3S-amino-1-benzyl-piperidin-2-one (240 mg, 0.54 mmol) and thereaction mixture was stirred at 30° C. for 48 h. Purification waseffected by removing excess amine and pentafluorophenol using scavengerresins. The pentafluorophenol was removed using a three fold excess(0.46 g, 1.62 mmol) of A-26 carbonate resin (3.5 mmol loading). Theresin was added to the reaction mixture and agitated for 3 h, afterwhich time it was removed by filtration. The excess amine was removedusing a five fold excess (2.7 g, 2.76 mmol) of methylisocyanatepolystyrene resin (1.2 mmol loading). The resin was added to thereaction mixture and agitated for 3 h, after which time it was removedby filtration and the solvent was removed in vacuo. Silica columnchromatography (dichloromethane:methanol 99:1 to 97:3) gave the puretitle compound (140 mg, 57%). 1H-NMR; δ (CDCl₃, rotamers) 8.15 (0.5H,s), 7.87 (0.5H, s), 7.36 (1 OH, m), 6.61 (1H, br.s), 4.98-4.47 (4H, m),4.28 (1H, m), 3.78 (1H, s), 3.22 (2H, m), 2.53 (2H, m), 1.82-1.29 (10H,m), 0.88 (3H, s). LRMS: +ve ion 466 [M+H]

Step E: 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoicAcid(1-benzyl-2-oxo-piperidin-3S-yl) amide

2R-[(Benzyloxy-amino-formyl)methyl]-hexanoic acid(1-benzyl-2-oxo-piperidin-3S-yl) amide (140 mg, 0.30 mmol) was dissolvedin ethanol (10 ml) and placed under a blanket of argon. Palladium oncharcoal (14 mg, 10% weight) was added and the mixture was stirredvigorously as hydrogen gas was bubbled through the system during 3 h.The flask was purged with argon before removing the catalyst byfiltration. The filtrate was concentrated under reduced pressure toprovide the title compound as a colourless oil (110 mg, 98%). ¹H-NMR; δ(CDCl₃, rotamers), 8.42 (0.5H, s) 7.80 (0.5H, s), 7.32 (5H, m),4.73-4.47 (2H, m), 4.24 (0.5H, m), 3.93 (1H, q), 3.61-3.34 (1H, s), 3.24(2H, m), 2.78-2.43 (2H, m), 2.23-1.24 (10H, m), 0.89 (3H, t, J=6.3 Hz).¹³C-NMR; δ (CDCl₃, rotamers), 174.3, 169.9, 136.1, 128.7, 127.8, 127.5,51.9, 50.9, 50.8, 47.1, 45.7, 29.3, 26.9, 22.5, 21.5, 20.7 and 13.8.LRMS: +ve ion 376 [M+H].

EXAMPLE 7 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2-oxo-azepan-3S-yl)-amide

The title compound was prepared in an analogous fashion to Example 6,from commercially available 3S-aminohexahydro-2-azepinone.

Colourless oil. ¹H-NMR; δ (CDCl₃, rotamers), 8.85 (0.5H, s) 7.79 (0.5H,s), 4.51 (1H, m), 3.71 (1H, m), 3.46-3.19 (3H, m), 2.86 (1H, t, J=8.5Hz), 1.98-1.66 (6H, m), 1.32-1.17 (6H, m), 0.89 (3H, t, J=6.6 Hz).¹³C-NMR; δ (CDCl₃, rotamers), 177.2, 172.3, 157.9, 53.7, 52.7, 45.6,42.7, 31.4, 30.1, 29.4, 29.0, 28.5, 23.2 and 14.3. LMRS: +ve ion 300[M+H].

EXAMPLE 8 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2,9-dioxo-1,8 diaza-cyclotetradec-3S-yl)-amide

The title compound was prepared by the method outlined in Scheme 4 andis described in detail below.

Step A: (6-Amino-2-benzyl-oxycarbonylamino-hexanoyl)-carbamicacid-9H-fluoren-9-yl Methylester, p-Toluenesulfonic Acid Salt

[5-Benzyloxycarbonylamino-6-(9H-fluoren-9-yl methoxycarbonylamino)-6-oxo-hexyl]-carbamic acid tert-butyl ester (0.6 g, 1mmol) was dissolved in dichloromethane (1 ml) and TFA (1 ml) was addeddropwise. The reaction mixture was stirred 3 h at room temperature.Para-toluenesulfonic acid was added and the mixture was stirred for afurther 30 minutes and concentrated in vacuo. The residue was trituratedwith diethyl ether and seeded. After 2 h at 4° C., the white solid wascollected by filtration. (0.49 g, 78%) ¹H-NMR: δ (CDCl₃) 7.77-7.27 (17H,m), 6.15 (1H, brs), 5.67 (1H, d, J=7.8 Hz), 5.06 (2H, m), 4.52-4.11 (5H,m), 2.75 (2H, br.s), 2.26 (3H, s) and 1.25-1.14 (6H, m), ¹³C-NMR; δ(CDCl₃) 172.1, 156.0, 143.4, 143.2, 141.2, 140.8, 136.2, 128.0, 124.8,119.8, 66.8, 66.6, 53.8, 46.7, 39.3, 31.3, 26.9, 21.8 and 21.2.

Step B:[2-Benzyloxycarbonylamino-6-(6-tert-butoxycarbonylamino-hexanoylamino)-hexanoyl]-carbamicacid 9H-fluoren-9-yl methyl ester.

To a solution of (6-amino-2-benzyl-oxycarbonylamino-hexanoyl)-carbamicacid 9H-fluoren-9-yl methylester, tosic acid salt (0.252 g, 0.4 mmol)and 6-tert-butoxycarbonylamino-hexanoic acid 4-nitro-phenyl ester (0.14g, 0.4 mmol) in dichloromethane (3 ml) was added imidazole (0.03 g, 0.44mmol). After stirring for 1 h at room temperature, a yellow colour wasobserved, then triethylamine (55 μl, 0.4 mmol) was added. The reactionmixture was stirred for 12h. The mixture was diluted and washed with 1Mhydrochloric acid (10 ml), 0.5M sodium carbonate (10 ml), brine (10 ml),dried over anhydrous magnesium sulphate and concentrated under reducedpressure. Flash column chromatography (dichloromethane-methanol, 9:1)gave a light foam (0.203 g, 76%). ¹H-NMR; δ (CDCl₃) 7.77-7.26 (13H, m),5.77 (1H, br.s), 5.60 (1H, d, J=8.0 Hz), 5.10 (2H, s), 4.65 (1H, br.s),4.49 (2H, m), 4.35 (1H, br.s), 4.19 (1H, m), 3.18(2H, m), 3.15 (2H, m),2.10 (2H, t, J=7.5 Hz), and 1.68-1.18 (21H, m).

Step C:[2-Benzyloxycarbonylamino-6-(6-tert-butoxycarbonylamino-hexanoylamino)-hexanoicAcid.

[2-Benzyloxycarbonylamino-6-(6-tert-butoxycarbonylamino-hexanoylamino)-hexanoyl]-carbamicacid 9H-fluoren-9-yl methyl ester was dissolved in dichloromethanecontaining 5% of diethylamine (2 ml) and stirred for 6 h at roomtemperature. The reaction mixture was concentrated in vacuo. Flashcolumn chromatography (dichloromethane-methanol-acetic acid 18:1:1) gavethe pure product (0.13 g, 87%). ¹H-NMR; δ (CDCl₃) 9.00-8.75 (1H, br.s),7.26 (5H, s), 6.31 (1H, br.s), 5.80 (1H, d, J=7.8 Hz), 5.12 (2H, s),4.85 (1H, br.s), 4.35 (1H, m), 3.11 (2H, m), 3.05 (2H, m), 2.17 (2H, t,J=7.4 Hz), and 1.95-1.20 (21H, m).

Step D:[2-Benzyloxycarbonylamino-6-(6-tert-butoxycarbonylamino-hexanoyfamino)-hexanoicAcid Pentafluoro-phenyl Ester.

A solution of[2-benzyloxycarbonylamino-6-(6-tert-butoxycarbonylamino-hexanoylamino)-hexanoicacid (0.13 g, 0.26 mmol) and pentafluorophenol (0.1 g, 0.54 mmol) indichloromethane (5 ml) was stirred and cooled in an ice bath during theaddition of EDC (55 mg, 0.29 mmol). The stirring was continued at roomtemperature for 3 h. The mixture was washed twice with 1M hydrochloricacid (10 ml), 0.5M sodium carbonate (10 ml), brine (10 ml), dried overanhydrous magnesium sulphate and concentrated under reduced pressure.The resulting gum was triturated with hexane to give an amorphous solid(0.13 g, 75%). ¹H-NMR; δ (CDCl₃) 7.32 (5H, s), 6.00 (2H, br.t and br.d),5.12 (2H, s), 4.63 (2H, m), 3.23 (2H, m), 3.03 (2H, m), 2.12 (2H, t,J=7.5 Hz), and 1.65-1.21 (21H, m).

Step E: 6-(6-Amino-hexanoylamino)-2-benzyloxycarbonylamino-hexanoic AcidPentafluoro-phenyl-ester, Trifluoroacetic Acid Salt.

[2-Benzyloxycarbonylamino-6-(6-tert-butoxycarbonylamino-hexanoylamino)-hexanoicacid pentafluorophenyl ester (130 mg, 0.2 mmol) was dissolved indichloromethane (3 ml) and TFA (0.5 ml) was added dropwise. The reactionmixture was stirred for 2 h at room temperature and concentrated invacuo to leave the crude title compound which was taken on to the nextstep without further purification.

Step F: (2,9-Dioxo-1,8-diaza-cyclotetradec-3S-yl)-carbamic Acid BenzylEster

6-(6-Amino-hexanoylaminoy2-benzyloxycarbonylamino-hexanoic acidpentafluorophenyl ester trifluoroacetic acid salt was dissolved inpyridine and heated to 80° C. (64 mg, 86%).

Step G: 3S-Amino-1 ,8-diaza-cyclotetradecane-2,9-dione

Palladium on charcoal (10%, 50 mg) was added to a stirred suspension of(2,9-dioxo-1,8-diaza-cyclotetradec-3-S-yl)-carbamic acid benzyl ester(500 mg, 1.33 mmol) in aceic acid-ethyl acetate 2:1 (6 ml) under argon.H₂ was bubbled through the suspension for 2 h and left under anatmosphere of hydrogen. The flask was purged with argon before thecatalyst was removed by filtration. The filtrate was concentrated underreduced pressure to provide the title compound as a yellow oil (254 mg,98%). ¹H-NMR; δ (CD₃OD) 3.69-3.40 (3H, m), 3.04-2.90 (2H, br.m),2.25-2.16 (2H, br.m), 1.94-1.12 (12H, br.m). LRMS: +ve ion 242 [M+H]

Step H: 2R-abenzycoxy-amino-formyl)-methyl]-hexanoic acid (2,9-dioxo-1,8-diaza -cyclotetradec-3S-yl)-amide

To a solution of 2R-[(benzyloxy-formyl-amino)-methyl]-hexylpentafluorophenyl ester (233 mg, 0.52 mmol) in DMF (10 ml) was added3S-amino-1.8-diaza-cyclotetradecane-2,9-dione (254 mg, 1.05 mmol) andthe reaction mixture was stirred at room temperature for 12 h. Thereaction mixture was concentrated in vacuo and the residue dissolved inmethanol-dichiloromethane 1:1(10 ml). Purification was effected byremoving excess amine and pentafluorophenol using scavenger resins. Thepentafluorophenol was removed using a three fold excess (0.44 g, 1.56mmol) of A-26 carbonate resin (3.5 mmol loading). The resin was added tothe reaction mixture and agitated for 3 h, after which time it wasremoved by filtration. The excess amine was removed using a five foldexcess (2.6 g, 2.60 mmol) of methylisocyanate polystyrene resin (1.2mmol loading). The resin was added to the reaction mixture and agitatedfor 3 h, after which time it was removed by filtration and the solventwas removed in vacuo. The resulting yellow solid was triturated withMeOH to give by filtration the title compound as a colourless solid (100mg, 38%).¹H-NMR; δ (CDCl₃, rotamers) 8.14 (0.5H, s), 7.87 (0.5H, s),7.40 (5H, m), 4.91 (2H, m), 4.28 (1H, m), 3.77-3.37 (3H, m), 2.95 (2H,m), 2.70 (H, m), 2.19 (2H, m), 1.69-1.27 (18H, m), 0.88 (3H, t, J=6.9Hz). LRMS: +ve ion 503 [M+H].

Step I: 2R-[(formyl-hydroxy-amino)-methyl]-hexanoicAcid(2,9-dioxo-1,8-diaza-cyclotetradec-3S-yl)-amide

2R-[(Benzyloxy-aminoformyl)-methyl]-hexanoic acid(2,9-dioxo-1,8-diaza-cyclotetradec-3S-yl)-amide (100 mg, 0.20 mmol) wasdissolved in methanol-chloroform 1:1 (10 ml) and placed under a blanketof argon. Palladium on charcoal (10 mg, 10% weight) was added and themixture was stirred vigorously as hydrogen gas was bubbled through thesystem for 2h. The flask was purged with argon before the catalyst wasremoved by filtration. The filtrate was concentrated under reducedpressure to provide the title compound as a colourless solid (80 mg,97%). ¹H-NMR; δ (CD₃OD, rotamers), 8.25 (0.4H, s), 8.15 (1H, m), 7.97(1H, m), 7.82 (0.6H, s), 4.27 (1H, m), 3.77-3.37 (4H, m), 2.99-2.85 (3H,m), 2.18 (2H, m), 1.66-1.31 (18H, m), 0.89 (3H, t, J=6.8 Hz). ¹³C-NMR; δ(CDCl₃, rotamers), 55.3, 53.9, 47.8, 40.2, 39.8, 36.9, 33.5, 31.2, 30.7,30.2, 26.9, 26.8, 24.1, 23.7 and 14.6. LMRS: +ve ion 425 [M+Na], 413[M+H].

EXAMPLE 9 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoicAcid(2-oxo-azacyclotridec-3R,S-yl)-amide

The title compound was prepared in an analogous fashion via steps H andI in scheme 6 from 3-amino-azacyclotnidecan-2one (Patent; Ajinomoto Co.Inc.; DE 1955038; Chem. Abstr.; EN; 73; 34829). Colourless solid.

¹H-NMR; δ (CDCl3, rotamers, mixture of diastereoisomers), 8.39 (0.3H,s), 8.38 (0.2H, s), 7.81 (0.3H, s), 7.79 (0.2H, s), 6.80-5.98 (2H, br.m)4.47 (1H, m), 3.76 (2H, m), 3.50 (1H, m), 2.96-2.65 (2H, m), 1.71-1.04(24H, m), 0.88 (3H, t, J=6.1 Hz). ¹³C-NMR; δ (CDCl₃, rotamers), 175.5,174.0, 53.6, 51.8, 44.8, 39.4, 31.9, 31.6, 29.4, 29.3, 28.9, 26.7, 26.3,24.7, 24.5, 23.8, 22.6 and 22.2. LMRS: +ve ion 384 [M+H].

EXAMPLE 10 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoicAcid[4-methyl-5-oxo-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-6S-yl]-amide

The compound was prepared according to the method described below (seealso Scheme 5):

Step A: 3-Amino-2S-benzyloxycarbonylamino-propionic Acid Methyl Ester,Hydrochloride

To a cooled solution (0° C.) of3-amino-2S-benzyloxycarbonylamino-propionic acid (1 g, 4.2 mmol) inmethanol (15 ml) was added dropwise thionyl chloride (0.36 ml, 5.04mmol). The reaction mixture was stirred for 4 hours at room temperatureand concentrated in vacuo to give a white crystal (1.2 g , 99%). ¹H-NMR;δ ((CD₃)₂SO): 8.25 (3H, br s), 7.92 (1H, d, J=8.3 Hz), 7.37 (5H, s),5.07 (2H, s), 4.484.39 (1H, m), 3.68(3H, s) and 3.24-3.06 (2H,br s).LRMS: +ve ion 253 [M+H].

Step B:2S-Benzytoxycarbonylamino-3-[2-(tert-butoxycarbonyl-methyl-amino)-ethylamino]-propionicAcid Methyl Ester, Hydrochloride

2-[N-(tert-Butoxycarbonyl)-N-methylamino)] acetaldehyde (S. Kato, H.Harada and T. Morie, J. Chem. Soc., Perkin Trans. 1, 1997, 3219 ) wasadded to a solution of 3-Amino-2S-benzyloxycarbonylamino-propionic acidmethyl ester, hydrochloride in methanol/acetic acid (99/1, 12 ml). Overa period of 45 minutes, sodium cyano borohydride was added portionwiseand the reaction mixture was stirred overnight at room temperature. Thelatter was diluted with ethyl acetate (150 ml) and a saturated solutionof sodium bicarbonate was added at 0° C. The organic layer was washedwith water (40 ml), brine (40 ml), dried over anhydrous magnesiumsulphate and concentrated in vacuo. The crude reaction mixture waspurified through flash chromatography (ethyl acetate) to give a clearoil (0.44 g, 63%). ¹H-NMR; δ (CDCl₃) 7.35 (5H, s), 5.88-5.71 (1H,br s),5.11 (2H, s), 4.41 (1H, m), 3.75 (3H, s), 3.26 (2H, m), 3.06-2.92 (2H,m), 2.83 (3H, s), 2.75 (2H, m). LRMS: +ve ion 410 [M+H]

Step C: 2S-Benzyloxycarbonylamino-3-(2-methylamino-ethylamino)-propionicAcid Methyl Ester, Dihydrochloride.

2S-Benzyloxycarbonylamino-3-[2-(tert-butoxycarbonyi-methyl-amino)ethylamino]-proprianicacid methyl ester, hydrochloride (200 mg, 0.49 mmol) was dissolved in

diethyl ether (3 ml) under a blanket of argon. To the reaction mixturecooled in an ice bath, hydrogen chloride 1M in diethyl ether (1.5 ml,1.5 mmol) was added. The reaction mixture was stirred overnight at roomtemperature and concentrated in vacuo to give the pure title compound(168 mg, 99%).¹H-NMR; δ (CDCl₃) 7.37 (5H, m), 5.14 (2H, s), 4.88 (1H,m), 3.77 (3H, s), 3.61-3.41 (6H, m), 2.77 (3H, s). LRMS: +ve ion 310[M+H]

Step D: 2S-Benzyloxycarbonylamino-3-(2-methylamino-ethylaminoypropionicAcid, Dihydrochloride.

2S-Benzyloxycarbonylamino-3-(2-methylamino-ethylaminopropionic acidmethyl ester, dihydrochloride (168 mg, 0.48 mmol) was dissolved inmethanol/ water (3/1, 4 ml) and lithium hydroxide (101 mg, 2.4 mmol) wasadded to the reaction mixture at 5° C. The reaction mixture was stirredat room temperature for 4 h, and then concentrated in vacuo. The residuewas dissolved in water (8 ml), acidified to pH =1 by means of 1M HCl andthe solvent was removed in vacuo to give the pure title compound (0.15g, 85%). ¹H-NMR; δ (CD₃0D) 7.38 (5H, m), 5.14 (2H, s), 4.58 (1H, m),3.65-3.45 (6H, m) and 2.77 (3H, s). LRMS: +ve ion 296 [M+H]

Step E: (1-Methyl-7-oxo-[1,4]diazepan-6S-yl)-carbamic Acid Benzyl Ester

2S-Benzyloxycarbonylamino-3-(2-methylamino-ethylamino)propionic acid,dihydrochloride (0.15 g, 0.4 mmol) was diluted in DMF (12 ml) and cooledin an ice bath. HOAt (8 mg, 0.06 mmol) and EDC (102 mg, 0.53 mmol) wereadded to the stirred solution and the stirring continued for 14 h. Thereaction mixture was concentrated in vacuo. Purification through flashchromatography (dichloromethane-methanol: 9515) gave the pure titlecompound (60 mg, 54%). ¹H-NMR; δ (CDCl₃) 7.35 (5H, s), 6.25 (1H, m),5.11 (2H, s), 4.48 (1H, m), 3.76-3.66 (2H, m), 3.27-3.05 (5H, m),2.97-2.66 (2H, m), 1.9 (2H, br s). LRMS: +ve ion 278 [M+H].

Step F:[4-Methyl-5-oxo-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-6S-yl]-CarbamicAcid Benzyl Ester

(1-Methyl-7-oxo-[1,4]diazepan-6S-yl)-carbamic acid benzyl ester (0.12 g, 0.43 mmol) was diluted in dry dichloromethane (5 ml) and cooled in anice bath. Triethylamine (60 μl, 0.43 mmol) and4-trifiuoromethyl-benzenesulfonyl chloride (105 mg, 0.43 mmol) wereadded to the stirred solution and the stirring continued for 5 h. Thereaction mixture was washed with water (5 ml), dried over anhydrousmagnesium sulphate and concentrated under reduced pressure to give awhite solid. (200 mg, 95%) ¹H-NMR; δ (CDCl₃) 8.02 (2H, d, J=8.0 Hz),7.80 (2H, d, J=8.0 Hz), 7.37 (5H, s), 6.22 (1H, m), 5.17 (2H, m), 4.53(1H, m), 4.25 (1H, m), 4.03 (1H, m), 3.63 (1H, m), 3.32 (1H,m), 3.01(3H, s), 2.82-2.60 (2H, m).

Step G:6S-Amino-4-methyl-l-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-5-oneHydrobromide

To a cooled solution (0° C.) of[4-Methyl-5-oxo-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-6S-yl]-carbamicacid benzyl ester (160 mg, 0.33 mmol) in acetic acid (2 ml), was addedhydrogen bromide (45% in acetic acid, 2 ml). The reaction mixture wasstirred for 3 hours at room temperature and 5 ml of diethyl ether wasadded to give a crystallisation. The white crystal was filtered off(0.14 g, 99%). ¹H-NMR: δ (CD₃OD) 8.01 (4H, m), 4.48 (1H, m) 4.08-3.78(3H, m), 3.57 (1H, m), 3.10-2.92 (5H, m). LMRS: +ve ion 352 [M+H]

Step H: 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid[4-methyl-5-oxo-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-6S-yl]-amide

To a solution of 2R-[(benzyloxy-formyl-amino)-methyl]-hexylpentafluorophenyl ester (107 mg, 0.24 mmol) in DMF (6 ml) were added6S-Amino-4-methyl-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-5-one, hydrobromide (125 mg, 0.29 mmol) and triethylamine (66μl, 0.48 mmol). The reaction mixture was stirred at room temperature for14 h. The solvent was removed under reduced pressure and the residue wasdissolved in dichloromethane. The triethylamine hydrobromide wasfiltered off, and the organic layer was concentrated in vacuo. Theresidue was taken-up in methanol (10ml). The excess of pentafluorophenoland amine were removed using scavenger resins, A-26 carbonate resin (3.5mmol loading, 0.2 g, 0.72 mmol) and methylisocyanate polystyrene resin(1.2 mmol loading, 1.0 g, 1.2 mmol) respectively. A purification throughflash chromatography (dichlormethane-methanol 99:1) gave the pure titlecompound (100 mg, 68%). ¹H-NMR; δ (CDCl₃, rotamers) 8.07 (3H, m), 7.81(2H, m), 7.38 (5H, br s), 7.01 (1H, br s), 5.014.68 (2H, m), 4.18 (1H,m), 3.89-3.48 (4H, m), 3.28 (1H, m), 3.01-2.88 (5H, m), 2.64-2.17 (2H,m), 1.74-1.25 (6H, m), 0.85 (3H, t, J=6.8 Hz). LRMS: +ve ion 635 [M+Na]

Step I: 2R-[(Formyl-hydroxy-aminoymethyl]-hexanoic acid[4-methyl-5-oxo-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-6S-yl]-amide

2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid[4-methyl-5-oxo-1-(4-trifluoromethyl-benzenesulfonyl)-[1,4]diazepan-6S-yl]-amide(100 mg, 0.16 mmol) was dissolved in ethanol (10 ml) and placed under ablanket of argon. Palladium on charcoal (10 mg, 10% weight) was addedand the reaction mixture was stirred vigorously while bubbling hydrogengas for 45 minutes. The reaction mixture was then stirred for 12 hoursunder an atmosphere of hydrogen. The flask was purged with argon beforeremoving the catalyst by filtration. The filtrate was concentrated underreduced pressure to provide the title compound as a colourless oil (75mg, 88%). ¹H-NMR; δ (CDCl₃, rotamers), 8.39 (0.25H, s), 8.04 (2H, d,J=8.2 Hz), 7.91 (0.75H, s), 7.82 (2H, d, J=8.1 Hz) 4.73 (1H, m), 3.87(2H, m), 3.51 (1H, m), 3.32 (1H, m), 3.02 (3H, m), 2.87 (1H, m) 2.75(1H,m), 1.84-1.23 (6H, m), 0.87 (3H, t, J=6.8 Hz). ¹³C-NMR; δ CDlC₃,rotamers), 172.9, 170.7, 141.0, 128.5, 128.3, 127.0, 126.9, 52.9, 52.5,50.2, 48.9, 47.3, 45.2, 36.9, 29.8, 29.7, 23.0 and 14.2. LRMS: +ve ion545 [M+Na].

What is claimed is:
 1. A compound selected from the group consisting of:2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4,7,7-trimethyl-5-oxo-[1,4]thiazepan-6R-yl) amide;2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(5,5-dimethyl-7-oxo-[1,4]oxathiepan-6-yl)-amide;2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(7,7-dimethyl-5-oxo-octahydro-8-thia-4a-aza-benzocyclohepten-6-yl)-amide;2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4-ethyl-7,7-dimethyl-5-oxo-[1,4]thiazapan-6-yl)-amide;2-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(4-propyl-7,7-dimethyl-5-oxo-[1,4]thiazapan-6-yl)-amide;2R-[(formyl-hydroxy-amino)-methyl]-hexanoic acid(1-benzyl-2-oxo-piperidin-3S-yl) amide;2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid(2-oxo-azepan-3S-yl)-amide; 2R-[(formyl-hydroxy-amino)-methyl]-hexanoicacid (2,9-dioxo-1,8 diaza-cyclotetradec- 3S-yl)-amide;2R-[(formyl-hydroxy-amino)-methyl]-hexanoic acid(2-oxo-azacyclotridec-3R,S-yl)-amide; and2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid[4-methyl-5-oxo-1-(4-trifluoromethyl-benzenesufonyl)-[1,4]diazepan6S-yl]-amide.2. A method for the treatment of bacterial infections in humans andnon-human mammals, which comprises administering to a subject sufferingsuch infection an antibacterially effective dose of a compound offormula (I) or a pharmaceutically or veterinarily acceptable salt,hydrate, or solvate thereof:

wherein: R₁ represents hydrogen, methyl, or trifluoromethyl; R₂represents a group R₁₀-(X)_(n)-(ALK)- wherein R₁₀ represents hydrogen, aC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, cycloalkyl, aryl, orheterocyclyl group, any of which may be unsubstituted or substituted by(C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino,halo, trifluoromethyl, cyano, nitro, -COOH, -CONH₂, -COOR^(A),-NHCOR^(A), -CONHR^(A), -NHR^(A), -NR^(A)R^(B), or -CONR^(A)R^(B)wherein R^(A) and R^(B) are independently a (C₁-C₆)alkyl group, and ALKrepresents a straight or branched divalent C₁-C₆ alkylene, C₂-C₆alkenylene, C₂-C₆ alkynylene radical, and may be interrupted by one ormore non-adjacent —NH—, —O— or —S— linkages, X represents —NH—, —O— or—S—, and n is 0 or 1; and R₃ and R₄, taken together with the carbonatoms to which they are respectively attached, form an optionallysubstituted saturated carbocyclic or heterocyclic ring of 5 to 16 atoms,which may be benz-fused or fused to a second optionally substitutedsaturated carbocyclic or heterocyclic ring of 5 to 16 atoms.
 3. A methodas claimed in claim 2 wherein R₁ is hydrogen.
 4. A method as claimed inclaim 3 wherein R₂ is C₁-C₆ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;phenyl(C₁-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)-optionally substituted in the phenyl ring; cycloalkyl(C₁-C₆ alkyl)-,cycloalkyl(C₃-C₆ alkenyl)- or cycloalkyl (C₃-C₆ alkynyl)- optionallysubstituted in the phenyl ring; heterocyclyl(C₁-C₆ alkyl)-,heterocyclyl(C₃-C₆ alkenyl)- or heterocyclyl (C₃-C₆ alkynyl)- optionallysubstituted in the heterocyclyl ring; or 4-phenylphenyl(C₁-C₆ alkyl)-,4-phenylphenyl(C₃-C₆ alkenyl)-, 4-phenylphenyl(C₃-C₆ alkynyl)-,4-heteroarylphenyl(C₁-C₆ alkyl)-, 4-heteroarylphenyl (C₃-C₆ alkenyl)-,4-heteroarylphenyl(C₃-C₆ alkynyl)-, optionally substituted in theterminal phenyl or heteroaryl ring.
 5. A method as claimed in claim 3wherein R₂ is methyl, ethyl, n- and iso-propyl, n- and iso-butyl,n-pentyl, iso-pentyl 3-methyl-but-1-yl, n-hexyl, n-heptyl, n-acetyl,n-octyl, methylsulfanylethyl, ethylsulfanylmethyl, 2-methoxyethyl,2-ethoxyethyl, 2-ethoxymethyl, 3-hydroxypropyl, allyl,3-phenylprop-3-en-1-yl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl,3-(2-chlorophenyl)prop-2-yn-1-yl, but2-yn-1-yl, cyclopentyl, cyclohexyl,cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl,cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, furan-2-ylmethyl,furan-3-methyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylmethyl,piperidinylmethyl, phenylpropyl, 4-chlorophenylpropyl,4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl,4-methylbenzyl, or 4-methoxybenzyl.
 6. A method as claimed in claim 3wherein R₂ is n-propyl, n-butyl, n-pentyl, benzyl or cyclopentylmethyl.7. A method as claimed in claim 3 wherein the ring formed by R₃ and R₄taken together with the carbon atoms to which they are respectivelyattached has from 5 to 8 ring atoms.
 8. A method as claimed in claim 3wherein the ring formed by R₃ and R₄ taken together with the carbonatoms to which they are respectively attached, has structure (III):


9. A method as claimed in claim 8 wherein R₈ is hydrogen, methyl, ethyl,n-propyl, cyclopentyl, phenyl or benzyl, and R₉ is hydrogen, isobutyl,phenyl or benzyl.